Method for selection of microorganism strains

Abstract

FIELD: biotechnology.SUBSTANCE: method for selecting probiotic bacteria based on an assessment of their sorption properties in relation to manganese, iron and copper is proposed, including cultivating strains of probiotic bacteria on a semi-synthetic nutrient medium of a given composition in a volume of 400 ml per strain with the addition of MnSO4, FeSO4, CuSO4 salts in a concentration of 0.031 to 0.061 mol. / l for 24–36 hours at a temperature of 37 °C, then centrifugation of the obtained biomass is carried out at 3000 rpm for 30 min, followed by removal of the supernatant and cell lysis is carried out using a 5% potassium hydroxide solution at a temperature of 98 °C for 20 minutes in a water bath, and the resulting lysates are analyzed by atomic absorption spectrophotometry, with probiotic bacteria that retain the ability to grow at salt concentrations of at least 0.031 mol. / l and demonstrate sorption activity in relation to microelement ions of at least 40% of the initial concentration considered the most promising.EFFECT: selection of probiotic bacteria for the prevention of hypomicroelementosis when administered orally.1 cl, 1 dwg, 1 ex

Description

[0001] The invention relates to biotechnology and can be used in biological and medical laboratories developing probiotic preparations for medical or veterinary use with predetermined properties. The method for selecting microorganisms for inclusion in probiotic preparations involves correcting microelement metabolism in the body receiving such probiotic preparations orally.

[0002] Thus, the claimed method is intended for use in biological and medical laboratories that address issues of creating new probiotic preparations with predetermined properties.

[0003] A known method for selecting microorganism strains for inclusion in probiotic preparations is based on the selection criteria based on the ability of microorganisms to reduce calcium and phosphorus losses in the tissues of laboratory animals [1]. This method evaluates the maximum compressive force of tubular bones of experimental animals on a mineral-deficient diet that received the preparations bifidumbacterin strain Bifidobacterium longum at a dose of 0.3 million μg / kg of live weight and sporobacterin strain Bacillus subtilis 534 at a dose of 50 million μg / kg. The studies were performed on 4 groups of 11-month-old male rats kept under conditions of a balanced diet according to the recommendations of the N.V. Sklifosovsky Research Institute. A mineral-deficient diet was achieved by drinking distilled water and feeding rice prepared in a special way (cooking polished rice for 15 minutes, then removing the broth and rinsing with distilled water).In order to prevent vitamin deficiency in rats, a multivitamin complex containing vitamins A, D, C, K, E, B1, B2, B3, B5, B6, B was introduced into the experimental diet. с , IN 12 .

[0004] The disadvantage of this method is that it does not take into account the ability of microorganisms to biosorb and tolerance to trace element salts, which limits its use in the creation of probiotic preparations capable of participating in the regulation of trace element metabolism.

[0005] Scientific literature has established that trace elements such as manganese, iron, cobalt, copper, and zinc are essential cofactors for enzymes that influence the metabolism of both microorganisms and macroorganisms. Microelement deficiencies lead to decreased enzymatic activity in the intestinal microbiota, which can negatively impact nutrient absorption and metabolism.

[0006] The objective of the invention is to select microorganisms for evaluating the sorption properties of monoculture probiotic strains E. coli M-17; L. acidophilus; B. longum; B. subtilis 534 in relation to manganese, iron and copper.

[0007] The technical result of the invention is the selection of microorganism strains for effective interaction with microelement ions for the prevention of hypomicroelementosis.

[0008] The specified technical result is achieved by selecting strains of microorganisms to evaluate the sorption properties of monoculture probiotic strains of E. coli M-17; L. acidophilus; B. longum; B. subtilis 534 in relation to manganese, iron and copper, including the cultivation of microorganism strains on a semi-synthetic nutrient medium in a volume of 400 ml per strain with the addition of MnSO4, FeSO4, CuSO4 salts at concentrations of 0.004 m / l, followed by incubation for 24-36 hours at a temperature of 37°C and centrifugation of the resulting biomass at 3000 rpm for 30 minutes, then the supernatant is removed and cell lysis is performed using a 5% potassium hydroxide solution at a temperature of 98°C for 20 minutes in a water bath, the resulting lysates are analyzed by atomic absorption spectrophotometry.

[0009] The essence of the invention is as follows.

[0010] For in vitro studies, the following probiotic preparations are used: Colibacterin (E. coli strain M-17), Lactobacterin (Lacobacillus acidophilus strain), Soya-bifidum (Bifidobacterium longum strain), Sporobacterin (Bacillus subtilis strain). A semi-synthetic medium for culturing test organisms without added microelements is used as a nutrient medium: casein - 100 g, 5% glucose - 200 ml, potassium dihydrogen phosphate - 85.94 g, calcium chloride - 212.5 g, sodium chloride - 7.25 g, magnesium sulfate - 13.57 g, distilled water - 1 l, to which MnSO4, FeSO4, CuSO4 salts are added at concentrations from 0.002 to 1.0 mol / l. Strain growth is assessed visually and by seeding on selective media. After incubation for 24-36 hours, the biomass is centrifuged at 3000 rpm for 30 minutes, the supernatant is removed and cell lysis is performed using a 5% potassium hydroxide solution at a temperature of 98 ° C for 20 minutes in a water bath.The obtained lysates are analyzed by atomic absorption spectrophotometry.

[0011] The most promising strains are those that maintain the ability to grow at salt concentrations of at least 0.031 mol / L and exhibit sorption activity for trace element ions of at least 40% of the initial concentration. This dual selection system—based on tolerance and sorption—allows us to identify strains with the potential to regulate trace element metabolism when administered orally.

[0012] Example of the method implementation

[0013] The following bacteria were selected as the objects of study: E. coli M-17; L. acidophilus; B. longum; B. subtilis 534 and salts of trace elements: manganese, iron and copper. To standardize the protocol, a serial dilution method was used with the addition of salts of trace elements MnSO4, FeSO4, CuSO4 in concentrations from 0.002 to 1.0 mol / L. By sequential transfer of 1:2, the minimum test concentration of 0.002 mol / L was obtained. A standard cell suspension with a density of 0.5 according to McFarland, optical density of 0.08-0.13 at 625 nm, was added to all tubes, the inoculum volume was 50 μl per 5 ml of medium. Incubation was carried out for 24-36 hours at a temperature of 37 ° C.

[0014] Growth was assessed visually by comparing turbidity with the control. No growth was recorded as an inhibitory effect; weak opalescence was recorded as a subinhibitory effect. A test tube without added salts served as the reference. Viability was confirmed by plating onto agar selective media.

[0015] After incubation, the biomass was collected by centrifugation, subjected to alkaline lysis, and the ion content was determined by atomic absorption spectrophotometry.

[0016] Table 1 presents a summary of the growth characteristics of four probiotic strains: E. coli M-17, L. acidophilus, B. longum and B. subtilis 534, when cultivated in the presence of three microelement salts: MnSO4, FeSO4 and CuSO4, in gradient ratios of 1:1, 1:2 and 1:4, concentrations of 0.002; 0.004 and 0.006 mol / L.

[0017] Thus, E. coli M-17 exhibits high tolerance to all three salts in all tested ranges, while L. acidophilus and B. longum demonstrate selective suppression at elevated concentrations of MnSO4 and CuSO4. The B. subtilis 534 strain shows stable growth at all FeSO4 concentrations. The optimal concentration for evaluating the sorption properties of monoculture probiotic strains E. coli M-17; L. acidophilus; B. longum; B. subtilis 534 in relation to manganese, iron and copper is 0.004 m / l, at a dilution of 1:2.

[0018] Sources of information:

[0019] 1. Patent for invention of the Russian Federation No. 2293118 Method for selecting microorganisms for their inclusion in probiotic strains / D.G. Deryabin, S.V. Notova, S.A. Miroshnikov, O.V. Kvan, Yu.B. Ivanov, S.V. Lebedev. Published 10.02.2007.

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